pOsHAK1：OsFLN2提高水稻的糖代谢水平和抗旱性

doi:10.13560/j.cnki.biotech.bull.1985.2021-1499

摘要/Abstract

摘要：

糖的合成和转运是植物响应干旱胁迫的关键过程。利用干旱诱导的钾转运蛋白基因OsHAK1的启动子驱动OsFLN2基因的异位表达,可以在干旱胁迫下改善水稻的糖代谢水平,进而促进根系生长。pOsHAK1：OsFLN2转基因植株叶片净光合速率、蔗糖磷酸合成酶活性、韧皮部蔗糖转运速率提高,叶片、根系中的蔗糖含量分别显著减少和增加。pOsHAK1：OsFLN2转基因株系的抗旱性优于野生型（WT）水稻,与WT相比,转基因植株脯氨酸积累量较高,而脂质过氧化水平较低。进一步研究发现,干旱处理的转基因株系中胁迫响应基因显著上调,而衰老相关基因的表达量低于WT。结果表明,通过pOsHAK1：OsFLN2的表达,促进糖分代谢,有利于提高水稻的抗旱性和在干旱胁迫下的生产力。

关键词: 水稻, 抗旱性, 糖代谢, 诱导型启动子

Abstract:

Sugar synthesis and transport are key processes in plants response to drought stress. Ectopic expression of OsFLN2 gene driven by the promoter of OsHAK1 encoding a drought-enhanced potassium transporter may improve the growth of root system by affecting sugar metabolism in rice under drought stress. The net photosynthetic rate and sucrose phosphate synthase activity in the leaves of the pOsHAK1：OsFLN2 transgenic seedlings increased,but the sucrose contents of the leaves and roots markedly decreased and increased,respectively,resulting from the enhanced sucrose translocation in phloem under drought conditions. pOsHAK1：OsFLN2 transgenic lines demonstrated better tolerance to drought stress than wild-type（WT）ones. In comparison to WT,the transgenic lines had higher accumulations of proline,and lower level of lipid peroxidation. Further investigation revealed that in response to the drought treatment,stress-responsive genes were notably up-regulated in the transgenic plants,however,senescence-associated genes were less abundantly transcribed than in WT. The results suggest that improving sugar metabolism via the expression of pOsHAK1：OsFLN2 may be beneficial to increasing drought tolerance and rice productivity under drought stress.

Key words: rice, drought tolerance, sugar metabolism, inducible promoter

陈光, 李佳, 杜瑞英, 王旭. pOsHAK1：OsFLN2提高水稻的糖代谢水平和抗旱性[J]. 生物技术通报, 2022, 38(8): 92-100.

CHEN Guang, LI Jia, DU Rui-ying, WANG Xu. pOsHAK1：OsFLN2 Expression Enhances the Drought Tolerance by Altering Sugar Metabolism in Rice[J]. Biotechnology Bulletin, 2022, 38(8): 92-100.

图/表 9

表1 荧光定量PCR所用引物

Table 1 Primers used for RT-qPCR assays

基因名称Gene name	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
UBQ5	CTCGCCGACTACAACATCCA	TCTTGGGCTTGGTGTACGTCTT
OsFLN2	CCGAATGGCTTCTCTTCTTCTC	GGCTCCTGATTGAGTTGGTACTACA
SGR	GCAATGTCGCCAAATGACG	GCTCACCACACTCATTCCCTAAAG
OsATG8a	AGCCCAGAAAAGGCCATCTT	CATCCTTGTTCTCTTCGTAGATTGC
OsSNAC1	GTCAAGACTGATTGGATCATGC	CCAATCATCCAACCTGAGAGA
OsMYB2	GAGCAGCGAGGAGGAGGT	TGTAGTTGACGAGCAGGAGGT

图1 干旱胁迫对水稻糖分代谢的影响水稻在正常IRRI营养液中生长6周,再用15% PEG处理10 d。A：净光合速率（Pn）;B：蔗糖磷酸合成酶（SPS）活性;C：叶片蔗糖外运速率（SER）;D-E：叶片（D）和根（E）中蔗糖含量。数值显示的是平均值±SE（n=5）,星号（*）表示正常和15% PEG处理之间在P<0.05水平下具有显著差异。FW：鲜重

Fig. 1 Effects of drought stress on sugar metabolism in rice Rice seedlings were growing in normal IRRI solution for 6 weeks,then treated with 15% PEG for 10 d. A：Net photosynthetic rate（Pn）. B：Sucrose phosphate synthase（SPS）activity. C：Rate of sucrose export（SER）from the leaf. D-E：Sucrose contents of the leaf（D）and root（E）. The values are means ± SE of 5 replicates. Significant differences between normal and 15% PEG treatment are indicated with asterisks（P < 0.05）. FW：Fresh weight

图2 表达载体的构建及水稻遗传转化过程 A：表达载体构建示意图;B-G：愈伤组织诱导（B,C）、选择（D）、分化（E,F）以及生根和炼苗（G）,B-D 标尺= 2 cm,E-G标尺 = 3 cm;H：GUS染色鉴定T0代转基因阳性株系,标尺= 1 cm

Fig. 2 Construction of expression vector and process of genetic transformation in rice A：Construction map of expression vector. B-G：Callus induction（B,C）,sele-ction of transformed calli（D）,shoot regeneration from resistant calli（E,F）,and hardening of transgenic plants（G）,bar in B-D=2 cm,bar in E-G=3 cm. H：Iden-tification of positive transgenic lines by GUS staining in T0 generation,bar=1 cm

图3 干旱胁迫下T1代pHAK1：FLN2转基因株系与WT的FLN2表达量和净光合速率差异分析水稻在正常IRRI营养液中生长2周,再用15% PEG处理7 d。A：地上部OsFLN2的RT-qPCR分析,UBQ5基因作为内参;B：净光合速率。将5个不含目的片段的分离株系合并为第2个对照,5个转基因阳性株系合并为pHAK1：FLN2。数值显示的是平均值±SE,不同的字母表示在P<0.05水平下具有显著差异

Fig. 3 Differential analysis of FLN2 expression and net photosynthetic rate between T1 generation of pHAK1：FLN2 transgenic lines and WT in response to drought stress Rice seedlings were growing in normal IRRI solution for 2 weeks,then treated with 15% PEG for 7 d. A：RT-qPCR analysis of endogenous OsFLN2 in the shoots of seedlings. UBQ5 was chosen as the reference sequence. B：Net photosynthetic rate. Results for the five null segregants were combined together as a second control（null segregant）,while five transgenic lines were combined together as pHAK1：FLN2. The values are means±SE. Significant differences at P < 0.05 are indicated with different letters

图4 干旱胁迫下pHAK1：FLN2转基因株系与WT的苗期生长差异分析 A：幼苗期植株在正常和15% PEG处理下的生长情况,标尺=5 cm;B-C：地上部（B）和根系（C）生物量（干重）。数值显示的是平均值±SE（n=5）,星号和ns分别表示WT和转基因株系之间在P<0.05水平下具有和没有显著差异。下同

Fig. 4 Seedling growth of pHAK1：FLN2 transgenic lines compared with WT in response to drought stress A：Growth performance of the seedlings under normal and 15% PEG treatment,bar = 5 cm. B-C：Shoot（B）and root（C）biomass（dry weight）. The values are means ± SE of 5 replicates. Significant differences between WT and transgenic lines are indicated with asterisks（P < 0.05）and ns indicates non-significant differences at that level of significance. The same below

图5 干旱胁迫下pHAK1：FLN2转基因株系与WT的糖代谢差异分析 A：蔗糖磷酸合成酶（SPS）活性;B：蔗糖外运速率（SER）;C-D：叶片（C）和根（D）中蔗糖含量。数值显示的是平均值±SE（n=5）

Fig. 5 Differential analysis of sugar metabolism between pHAK1：FLN2 transgenic lines and WT in response to drought stress A：Sucrose phosphate synthase（SPS）activity. B：Rate of sucrose export（SER）from the leaf. C-D：Sucrose contents of the leaf（C）and root（D）.The values are means±SE of 5 replicates

图6 干旱胁迫下pHAK1：FLN2转基因株系与WT的根系构型差异分析 A：总根长;B：根表面积。数值显示的是平均值±SE（n=5）

Fig. 6 Differential analysis of root system architecture be-tween pHAK1：FLN2 transgenic lines and WT in response to drought stress A：Total root length. B：Root surface area. The values are means ± SE of 5 replicates

图7 干旱胁迫下pHAK1：FLN2转基因株系与WT的保水能力和脂质过氧化程度差异分析 A：相对含水量;B：叶片失水率;C：电解质渗透率;D：脯氨酸含量。数值显示的是平均值±SE（n=5）

Fig. 7 Differential analysis of water-content ability and lipid peroxidation between pHAK1：FLN2 trans-genic lines and WT in response to drought stress A：Relative water content. B：Water loss rate. C：Relative electrolyte leakage. D：Proline content. The values are means±SE of 5 replicates

图8 干旱胁迫下pHAK1：FLN2转基因株系与WT中衰老和胁迫响应相关基因的表达差异分析定量检测的基因有SGR（A）,ATG8a（B）,SNAC1（C）和MYB2（D）。UBQ5基因作为内参。数值显示的是平均值±SE（n=3）

Fig. 8 Differential analysis of expressions of senescence-associated genes and stress-responsive genes between pHAK1：FLN2 transgenic lines and WT in response to drought stress The genes assayed are（A）SGR,（B）ATG8a,（C）SNAC1 and（D）MYB2. UBQ5 is chosen as the reference gene. The values are means±SE of 3 replicates

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